Pascual Martínez-Peinado

Effects of pomegranate juice in circulating parameters, cytokines, and oxidative stress markers in endurance-based athletes: A randomized controlled trial

OBJECTIVEnThe aim of the present study was to assess the effects of pomegranate juice on the level of oxidative stress in the blood of endurance-based athletes. Pomegranate juice is rich in polyphenols, conferring it a higher antioxidant capacity than other beverages with polyphenolic antioxidants.nnnMETHODSnA randomized double-blind, multicenter trial was performed in athletes from three different sport clubs located in southeastern of Spain. Plasma oxidative stress markers (protein carbonyls and malondialdehyde [MDA]) as well as C-reactive protein and sE-selectin were measured. Thirty-one athletes participated in the study. Participants were divided into three groups. The first group was supplemented with 200xa0mL/d pomegranate juice (PJ; nxa0=xa010) over a 21-d period, the second with 200xa0mL/d pomegranate juice diluted 1:1 with water (PJD; nxa0=xa011), and a control group that did not consume pomegranate juice (C; nxa0=xa010). Nine athletes were excluded due to protocol violations (nxa0=xa04 in the PJ group and nxa0=xa05 in the PJD group) because they did not observe the 24xa0h of rest before the last blood test.nnnRESULTSnThe control group increased levels of carbonyls (+0.7xa0±xa00.3xa0nmols/mg protein) and MDA (+3.2xa0±xa01.0xa0nmols/g protein), whereas the PJ and PJD groups maintained or decreased their levels, respectively. On the other hand, lactate levels increased in the PJ group (from 10.3 at day 0 to 21.2xa0mg/dL at day 22). A nonsignificant decrease was detected in sE-selectin and C-reactive protein in the groups consuming pomegranate juice.nnnCONCLUSIONnConsumption of pomegranate juice over a 21-d period improved MDA levels and carbonyls, and thus decreased the oxidative damage caused by exercise.

Carbon and nitrogen limitation increase chitosan antifungal activity in Neurospora crassa and fungal human pathogens

Chitosan permeabilizes plasma membrane and kills sensitive filamentous fungi and yeast. Membrane fluidity and cell energy determine chitosan sensitivity in fungi. A five-fold reduction of both glucose (main carbon (C) source) and nitrogen (N) increased 2-fold Neurospora crassa sensitivity to chitosan. We linked this increase with production of intracellular reactive oxygen species (ROS) and plasma membrane permeabilization. Releasing N. crassa from nutrient limitation reduced chitosan antifungal activity in spite of high ROS intracellular levels. With lactate instead of glucose, C and N limitation increased N. crassa sensitivity to chitosan further (4-fold) than what glucose did. Nutrient limitation also increased sensitivity of filamentous fungi and yeast human pathogens to chitosan. For Fusarium proliferatum, lowering 100-fold C and N content in the growth medium, increased 16-fold chitosan sensitivity. Similar results were found for Candida spp. (including fluconazole resistant strains) and Cryptococcus spp. Severe C and N limitation increased chitosan antifungal activity for all pathogens tested. Chitosan at 100 μg ml(-1) was lethal for most fungal human pathogens tested but non-toxic to HEK293 and COS7 mammalian cell lines. Besides, chitosan increased 90% survival of Galleria mellonella larvae infected with C. albicans. These results are of paramount for developing chitosan as antifungal.

Effects of a Lifestyle Program on Vascular Reactivity in Macro- and Microcirculation in Severely Obese Adolescents

CONTEXT AND OBJECTIVEnThis study aimed to comprehensively assess the macro- and microcirculation of severely obese adolescents (SOA) and normal-weight counterparts and to determine the longitudinal effects of weight loss on vascular function in SOA. DESIGN, SETTING, PARTICIPANTS, AND OUTCOME MEASURES: Seventeen SOA (body mass index z-score = 4.22 ± 0.73) and 19 puberty-matched normal-weight counterparts (body mass index z-score = -0.02 ± 1.04) were included. The SOA participated in a 4 month weight loss program. Brachial artery flow-mediated dilation and response to sublingual nitrate (nitrate-mediated dilation [NMD]) were assessed by high-resolution ultrasound. Microvascular reactivity was evaluated by laser Doppler flowmetry in response to NMD, iontophoresis of acetylcholine and sodium nitroprusside, and local hyperthermia. Plasma insulin, leptin, resistin, C-reactive protein, myeloperoxidase, and tissue plasminogen activator were measured.nnnRESULTSnAt baseline, SOA had similar flow-mediated dilation and impaired NMD in the brachial artery compared to normal-weight adolescents. Similarly, peak responses to acetylcholine and sodium nitroprusside iontophoresis and to local hyperthermia were unaltered, whereas cutaneous blood flow after NMD was lower in the forearm microcirculation of SOA. All plasma measurements were significantly higher in SOA. After the 4-month program, SOA presented a weight reduction of 7.4 ± 3.1%, but neither brachial artery nor microvascular reactivity variables were improved. Significant decreases were detected in plasma leptin, myeloperoxidase, and tissue plasminogen activator.nnnCONCLUSIONSnMacro- and microvascular endothelial function are preserved in adolescents with severe obesity. Conversely, weight loss does not improve their impaired smooth muscle response to exogenous organic nitrate in both vascular beds, despite reducing plasma markers adversely related to vascular homeostasis.

Determination of aflatoxin M1 in milk samples by means of an inductively coupled plasma mass spectrometry-based immunoassay

An inductively coupled plasma mass spectrometry (ICP-MS)-based immunoassay has been developed to quantify aflatoxin M1 (AFM1) at ultra-trace levels in milk samples. AFM1 detection is carried out by means of a competitive immunoassay using secondary biotinylated antibodies and streptavidin-conjugated Au nanoparticles. After acid addition, nanoparticles are decomposed and Au signal is registered by means of ICP-MS. Results demonstrate that, under optimum conditions, the limit of detection of the immunoassay (0.005μgkg-1) is low enough to quantify AFM1 according to current international policies (including the more restrictive European one). Method accuracy and precision was checked by analyzing an AFM1 certified reference material and different milk samples spiked with known amounts of AFM1. AFM1 recovery values range from 80% to 102% whereas inter-assay and intra-assay precision are lower than 15%. Finally, this immunoassay methodology affords a higher dynamic working range (0.012-2.5μgkg-1) than other immunoassay methodologies described in the literature.

Differences of Clonogenic Mesenchymal Stem Cells on Immunomodulation of Lymphocyte Subsets

Mesenchymal stem cells (MSC) are a widely used population in cell therapy for their ability to differentiate into distinct tissues and more lately, for their immunomodulatory properties. However, the use of heterogeneous populations could be responsible for the nondesired outcomes reflected in the literature. Here, we analyse the different capacities of five one-cell-derived MSC clones to exert their immunomodulation ex vivo. We assessed proliferation assays in cocultures of MSC clones and purified cluster of differentiation (CD)3+, CD4+, or CD8+ lymphocytes; analysed the regulatory T (Treg) cells fold change rate; determined the effects on viability of peripheral blood mononuclear cells (PBMC); and also measured the coculture cytokine profiles (Th1/Th2). Conditioned media (CM) of different clones were also used to perform both proliferation assays and to analyse Treg fold change. The five clones analysed in this work were able to generate heterogeneous environments. Different clones inhibited proliferation of CD3+ and CD4+ lymphocytes, with different intensities. Surprisingly, all clones promoted proliferation of CD8+ lymphocytes. Different MSC clones and their CM were able to increase the number of Treg with different intensities. Finally, different clones also promoted different effects on the viability of PBMC treated with ultraviolet light. Considering all these data together, it seems that different clones, even from the same donor, can promote a wide spectrum of responses from anti-inflammatory to proinflammatory character. This fact may be important to standardise the design of personalized cell therapy protocols, thus diminishing the aforementioned undesired outcomes existing nowadays in this type of therapies.

CD44 induces FOXP3 expression and is related with favorable outcome in breast carcinoma

We studied the relationship between CD44 and Forkhead box P3 (FOXP3) gene expression in cell lines and breast carcinomas and their association with clinicopathological variables and patient outcome. We assessed messenger RNA (mRNA) expression of CD44 and FOXP3 by quantitative real-time PCR and determined the number of FOXP3+ Tregs by immunohistochemistry in 264 breast cancer specimens. CD44 was stimulated with hyaluronan treatment, and the accompanying changes in FOXP3 mRNA expression in breast cancer cell lines representing breast cancer subtype were assessed. We found that lower CD44 expression correlated with the presence of necrosis, lymph-vascular invasion, grade 3 tumors, and aggressive phenotype (HER2 and basal-like). FOXP3 mRNA correlated positively with CD44 mRNA expression and Treg content. Moreover, stimulation of CD44 expression by hyaluronan in cell lines increased FOXP3 expression, which supports that their regulation is associated. Survival analysis revealed that low CD44 expression is associated with higher frequency of recurrence. Our findings indicate that CD44 has a regulatory role in FOXP3 expression and is associated with good prognostic factors in breast cancer.

Effect of a 2000-m running test on antioxidant and cytokine response in plasma and circulating cells

Exercise intensity usually correlates with increased oxidative stress and enhanced cytokine production. However, it is unknown if all types of exercise that induce muscle damage can cause a parallel response in the oxidation balance and cytokine production. To this end, the effect of a 2000-m running test in a group of volunteers that regularly train in aerobic routines was studied. Different circulating parameters were measured, oxidative stress markers (protein carbonyls and malondialdehyde), antioxidant enzyme activity, and cytokine levels in plasma as well as in the main circulating cells of blood samples obtained in basal conditions and after test execution. As a result, the test caused muscle damage evidenced by an increase in circulating creatine kinase and myoglobin. This was accompanied by an increase in protein carbonyls in plasma and peripheral blood mononuclear cells. Activities of antioxidant enzymes (catalase, glutathione peroxidase and reductase, superoxide dismutase) were elevated in peripheral blood mononuclear cells, neutrophils, and erythrocytes after the test. Regarding cytokine production, interleukin-6, interleukin-8, interleukin-10, and tumor necrosis factor-α exhibited no significant changes after the test. Results suggest that this short but intense running exercise (2000xa0m) can induce muscle damage and elicit a good balance between oxidant/antioxidant responses with no changes in the circulating concentration of pro-inflammatory cytokines.